This invention relates to compounds for use in methods of shortening the time to alleviation of influenza symptoms; crystals of certain compounds; and pharmaceutical compositions including thereof.

Influenza is an acute respiratory infectious disease caused by infection with an influenza virus. In Japan, millions of influenza-like patients are reported every winter, and influenza is accompanied with high morbidity and mortality. Influenza is a particularly important disease in a high risk population such as baby and elderly, a complication rate with pneumonia is high in elderly, and death with influenza is occupied with elderly in many cases.

As anti-influenza drugs, Symmetrel (trade name: Amantadine) and Flumadine (trade name: Rimantadine) which inhibit the denucleation process of a virus, and Oseltamivir (trade name: Tamiflu) and Zanamivir (trade name: Relenza) which are neuraminidase inhibitors suppressing virus budding and release from a cell are known. However, since problems of appearances of resistant strains and side effects, and worldwide epidemic of a new-type influenza virus having high pathogenicity and mortality are feared, development of an anti-influenza drug having a novel mechanism has been desired.

Since a cap-dependent endonuclease which is an influenza virus-derived enzyme is essential for virus proliferation, and has the virus-specific enzymatic activity which is not possessed by a host, it is believed that the endonuclease is suitable for a target of an anti-influenza drug. The cap-dependent endonuclease of an influenza virus has a host mRNA precursor as a substrate, and has the endonuclease activity of producing a fragment of 9 to 13 bases including a cap structure (not including the number of bases of the cap structure). This fragment functions as a primer of a virus RNA polymerase, and is used in synthesizing mRNA encoding a virus protein. That is, it is believed that a substance which inhibits the cap-dependent endonuclease inhibits synthesis of a virus protein by inhibiting synthesis of virus mRNA and, as a result, inhibits virus proliferation.

As the substance which inhibits the cap-dependent endonuclease, flutimide (Patent Document 1 and Non-Patent Documents 1 and 2), 4-substituted 2,4-dioxobutanoic acid (Patent Document 2 and Non-Patent Documents 3 and 4), the compounds described in Patent Documents 3 to 12 and the like have been reported, but they have not yet led to clinical use as anti-influenza drugs. Patent Documents 9 and 12 describe compounds having a similar structure to that used in the present invention, but does not describe the compounds used in the present invention. Also, Patent Documents 13 to 15 describe compounds having a similar structure to that used in the present invention as a compound having integrase inhibitory activity, however, the documents do not describe cap-dependent endonuclease. In addition, Patent Document 16 and 17 describes an invention relating to compounds having a similar structure to that used in the present invention as a compound having cap-dependent endonuclease inhibitory activity, which has been filed by the applicants, but does not describe the compounds used in the present invention.

• Patent Document 1: GB2280435
• Patent Document 2: US5475109
• Patent Document 3: US20130090300
• Patent Document 4: WO2013/057251
• Patent Document 5: WO2013/174930
• Patent Document 6: WO2014/023691
• Patent Document 7: WO2014/043252
• Patent Document 8: WO2014/074926
• Patent Document 9: WO2014/108406
• Patent Document 10: WO2014/108407
• Patent Document 11: WO2014/108408
• Patent Document 12: WO2015/038655
• Patent Document 13: WO2005/016927
• Patent Document 14: WO2006/066414
• Patent Document 15: WO2007/049675
• Patent Document 16: WO2010/147068
• Patent Document 17: WO2012/039414

• Non-Patent Document 1: Tetrahedron Lett 1995, 36(12), 2005
• Non-Patent Document 2: Tetrahedron Lett 1995, 36(12), 2009
• Non-Patent Document 3: Antimicrobial Agents And Chemotherapy, Dec. 1994, p.2827-2837
• Non-Patent Document 4: Antimicrobial Agents And Chemotherapy, May 1996, p.1304-1307

• EP 3 473 629 was published on 28 December 2017 as WO 2017/221869. It describes certain compounds for treating influenza; crystals of such compounds and methods for producing such compounds. Certain intermediates are also discussed.
• WO 2017/104691 was published on 22 June 2017. It describes combination therapies for treatment of influenza.
• WO 2016/175224 was published on 3 November 2016. It describes similar compounds for treating influenza.
• PAULINA KOSZALKA et al, "Influenza antivirals currently in late-phase clinical trial", INFLUENZA AND OTHER RESPIRATORY VIRUSES, vol. 11, no. 3, 1 May 2017, pages 240-246 describes various influenza-targeting antiviral drugs which were at the time in clinical trials.

An object of the present invention is to provide compounds for use in methods of shortening the time to alleviation of influenza symptoms; crystals of certain compounds; and pharmaceutical compositions including thereof.

The present invention provides inventions set out in the appended claims.

The compound used in the present invention is a prodrug, and the prodrug becomes a parent compound having an inhibitory activity on cap-dependent endonuclease in vivo after administration, thus is effective as a therapeutic agent and/or preventive agent for influenza virus infectious disease.

• [Figure 1] Figure 1 is a result of measuring the plasma concentration of Compound III-2, after oral administration of prodrug Compound II-6, the parent compound of which is Compound III-2, to rat under non-fasting conditions.
• [Figure 2] Figure 2 is a result of measuring the plasma concentration of Compound II-6, after oral administration of prodrug Compound II-6, the parent compound of which is Compound III-2, to rat under non-fasting conditions.
• [Figure 3] Figure 3 is powder X-ray diffraction data of I-form crystals of Compound II-6. The horizontal axis indicates 2θ, and the vertical axis indicates intensity.
• [Figure 4] Figure 4 is powder X-ray diffraction data of II-form crystals of Compound II-6.
• [Figure 5] Figure 5 is powder X-ray diffraction data of III-form crystals of Compound II-6.

The meaning of each term used in the present description is explained below. Each term is used in a unified sense, and is used in the same sense when used alone, or when used in combination of other term.

The term of "consisting of" means having only components.

The term of "comprising" means not restricting with components and not excluding undescribed factors.

The compounds used in the present invention are characterized in that the compounds isolated by optical resolution of tricyclic compounds substituted by the other tricyclic group improve cap-dependent endonuclease inhibitory activity.

The compounds used in the present invention are also characterized in that the compounds are efficiently absorbed into the body after administration (for example, oral administration), and showing high efficacy by introducing a group to form a prodrug.

One or more hydrogen, carbon and/or other atoms in the compounds used in the present invention may be replaced with isotopes of hydrogen, carbon and/or other atoms respectively. Examples of isotopes include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine and chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, ¹²³I and ³⁶Cl respectively. The compounds used in the present invention include compounds replaced with these isotopes. The compounds replaced with the above isotopes are useful as medicines and include all of radiolabeled compounds used in the present invention. A "method of radiolabeling" in the manufacture of the "radiolabeled compounds" is encompassed by the present invention, and the "radiolabeled compounds" are useful for studies on metabolized drug pharmacokinetics, studies on binding assay and/or diagnostic tools.

A radiolabeled compound used in the present invention can be prepared using well-known methods in this field of the invention. For example, a tritium-labeled compound used in the present invention can be prepared by introducing a tritium to a certain compound used in the present invention, through a catalytic dehalogenation reaction using a tritium. This method comprises reacting with an appropriately-halogenated precursor of the compound used in the present invention with tritium gas in the presence of an appropriate catalyst, such as Pd/C, and in the presence or absent of a base. The other appropriate method of preparing a tritium-labeled compound can be referred to "Isotopes in the Physical and Biomedical Sciences, Vol. 1, Labeled Compounds (Part A), Chapter 6 (1987)". A ¹⁴C-labeled compound can be prepared by using a raw material having ¹⁴C.

The pharmaceutically acceptable salts of the compounds used in the present invention include, for example, salts with alkaline metal (e.g., lithium, sodium, potassium or the like), alkaline earth metal (e.g., calcium, barium or the like), magnesium, transition metal (e.g., zinc, iron or the like), ammonia, organic bases (e.g., trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, meglumine, ethylenediamine, pyridine, picoline, quinoline or the like) or amino acids, or salts with inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, carbonic acid, hydrobromic acid, phosphoric acid, hydroiodic acid or the like) or organic acids (e.g., formic acid, acetic acid, propionic acid, trifluoroacetic acid, citric acid, lactic acid, tartaric acid, oxalic acid, maleic acid, fumaric acid, mandelic acid, glutaric acid, malic acid, benzoic acid, phthalic acid, ascorbic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or the like). Especially, salts with hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, methanesulfonic acid and the like are included. These salts can be formed by the usual methods.

The compounds used in the present invention or its pharmaceutically acceptable salts may form solvates (e.g., hydrates or the like) and/or crystal polymorphs. "Solvates" may be those wherein any numbers of solvent molecules (e.g., water molecules or the like) are coordinated with the compounds used in the present invention. When the compounds used in the present invention or its pharmaceutically acceptable salts are allowed to stand in the atmosphere, the compounds may absorb water, resulting in attachment of adsorbed water or formation of hydrates. Recrystallization of the compounds used in the present invention or its pharmaceutically acceptable salts may produce crystal polymorphs.

A general method for producing the compound used in the present invention will be exemplified below. As to the extraction and purification, treatment which is performed in a normal experiment of organic chemistry may be conducted.

Synthesis of the compound used in the present invention can be carried out referring to the procedures known in the art.

As a raw material compound, commercially available compounds, compounds described in the present description, compounds described in the references cited in the present description, and other known compounds can be utilized.

When one wants to obtain a salt of the compound of the present invention, in the case where the compound used in the present invention is obtained in a form of a salt, it may be purified as it is and, in the case where the compound used in the present invention is obtained in a free form, a salt may be formed by a normal method by dissolving or suspending the compound in a suitable organic solvent, and adding an acid or a base.

In addition, the compound used in the present invention and a pharmaceutically acceptable salt thereof are present in a form of adducts with water or various solvents (hydrate or solvate) in some cases, and these adducts are used in the present invention.

The compounds used in the present invention are prodrugs for compounds which have cap-dependent endonuclease inhibitory activity and are useful as therapeutic or preventive agents for influenza.

The compounds used in the present invention are useful for symptoms and/or diseases which are induced by influenza virus. For example, they are useful for treating, preventing, and/or improving symptoms of, cold-like symptoms accompanying fever, cihll, headache, muscular or joint pain, fatigue etc., airway inflammation symptoms such as sore throat, nasal secretion, nasal congestion, cough, sputum etc., gastrointestinal symptoms such as abdominal pain, vomitus, diarrhea etc. and, further, complications accompanying secondary infection such as acute encephalopathy and pneumonia. That is to say, the compounds used in the present invention are useful for treating and/or preventing influenza virus infectious diseases.

The compounds used in the present invention are effective for shortening time to alleviation of influenza symptoms. For example, they can shorten the time to alleviation of influenza symptoms about 20 to 40 hours, preferably about 25 to 30 hours. Specifically, they can shorten the times until "cough", "sore throat", "headache", "nasal congestion", "feverishness or chills", "muscular or joint pain", and "fatigue" are alleviated. In particular, they are useful for shortening the times until "nasal congestion", "muscular or joint pain", "fatigue", "feverishness or chills", and "headache" are alleviated. Further, they are useful for shortening the times until "nasal congestion" and "muscular or joint pain" are alleviated.

Furthermore, since the compounds used in the present invention reduces the influenza virus in the body in a short period of time, it can be an excellent pharmaceutical useful for treating and/or preventing influenza virus infectious diseases. After the administration of the compounds used in the present invention, the effect of decreasing the influenza virus amount in the body is observed within 72 hours, preferably within 48 hours, more preferably within 24 hours, and it is expected an earlier therapeutic effect is obtained as compared with other drugs.

Moreover, the compounds used in the present invention have usefulness as medicines.

For example, since the compounds (prodrugs) used in the present invention have advantages that oral absorbability is high, good bioavailability is exhibited, good clearance is exhibited, and pulmonary transitivity is high, they can be excellent medicaments.

Since the compounds (parent compounds) formed from the compounds (prodrugs) used in the present invention have the effects such as high inhibitory activity on cap structure-dependent endonuclease, and high selectivity due to a virus-specific enzyme, they can be medicaments having reduced side effects.

Further, the compounds used in the present invention also have advantages that metabolism stability is high, solubility is high, oral absorbability is high, good bioavailability is exhibited, good clearance is exhibited, pulmonary transitivity is high, a half life is long, a non-protein binding rate is high, hERG channel inhibition is low, CYP inhibition is low, CPE (CytoPathic Effect) inhibiting effect is recognized, and/or negative effects are exhibited in a phototoxicity test, an Ames test and a gene toxicity test, or toxicity such as liver damage is not caused. Therefore, the pharmaceutical composition of the present invention can be an excellent medicament.

The compounds used in the present invention can be administered orally or parenterally. In the case of oral administration, the compounds used in the present invention can be also used as a normal preparation, for example, as any dosage form of solid preparations such as tablets, powders, granules, capsules etc.; solutions; oleaginous suspensions; or liquid preparations such as syrups or elixirs etc. In the case of parenteral administration, the compounds used in the present invention can be used as aqueous or oleaginous suspension injectables, or nose drops. Upon preparation of them, conventional excipients, binders, lubricants, aqueous solvents, oleaginous solvents, emulsifiers, suspending agents, preservatives, stabilizers etc. can be arbitrarily used. The pharmaceutical composition of the present invention can be produced by combining (for example, mixing) a therapeutically and/or prophylactically effective amount of the compound used in the present invention with pharmaceutically acceptable carriers or diluents. The compounds used in the present invention can be suitably used as oral preparations because of their high oral absorbability.

A dose of the compounds used in the present invention is different depending on an administration method, an age, a weight and the state of a patient, and a kind of a disease.
According to the present invention, the compound is administered orally in a dose of 10 to 80 mg/day for an adult, if necessary, in divided doses. In another embodiment, in the case of adults, about 40 mg or 80 mg may be administered in a single dose. According to the present invention, the compound is administered orally in a single dose of 5 to 40mg in the case of children depending on body weight . In addition, in the case of parenteral administration (not according to the present invention), about 0.01 mg to 1000 mg, preferably about 0.05 mg to 500 mg, or about 1 mg to 80 mg for adult per day is administered. The dose may be administered once daily or may be divided into multiple doses per day.

The compounds used in the present invention can be used in combination with other drugs or the like (hereinafter referred to as combination drugs) to increase the activity of the compounds, reduce the dose of the compounds, or the like. In the case of treating influenza, the compounds can be used combined with or in a coupled formulation with neuraminidase inhibitor (e.g., Oseltamivir, Zanamivir, Peramivir, Inabiru and the like); RNA-dependent RNA polymerase inhibitor (e.g., Favipiravir); M2 protein inhibitor (e.g., Amantadine); PB2 Cap binding inhibitor (e.g., VX-787); anti-HA antibody (e.g., MHAA4549A); Immune agonists (e.g., Nitazoxanide) are also possible. In this case, the timing of administration for a compound used in the present invention and the combination drug is not limited. They can be administered to the subjects to be treated, at a time or at different times. Furthermore, the combination drug with a compound used in the present invention can be administered as two or more formulations independently comprising each active ingredient or a single formulation comprising each active ingredient.

The dose for combination drugs may be appropriately selected in reference to the clinical dose. The compounding ratio of the compounds used in the present invention and co-administered drugs may be appropriately selected depending on the subject to be treated, administration route, disease to be treated, symptoms, combination of the drugs and the like. For administration in humans, for example, 1 part by weight of the compounds used in the present invention may be used in combination with 0.01 to 100 parts by weight of co-administered drugs.

The present invention will be explained in more detail below by way of Examples, Intermediate Synthesis Examples, as well as Test Examples of the present invention, but the present invention is not limited to them.

The NMR analysis obtained in each example was carried out in 300 MHz, and was measured using DMSO-d₆, CDCl₃.

The term RT represents a retention time at LC/MS: liquid chromatography/mass spectrometry, and was measured under the following conditions.

1. (1) Column: ACQUITY UPLC (Registered trademark) BEH C18 (1.7µm i.d.2.1x50mm) (Waters)
   • Flow rate: 0.8 mL/min
   • UV detection wavelength: 254nm
   • Mobile phase: [A]: a 0.1% formic acid-containing aqueous solution, [B]: a 0.1% formic acid-containing acetonitrile solution
   • Gradient: a linear gradient of 5% to 100% solvent [B] was carried out in 3.5 minutes, and 100% solvent [B] was kept for 0.5 minutes.
2. (2) Column: Shim-pack XR-ODS (2.2pm, i.d.50x3.0mm) (Shimadzu)
   • Flow rate: 1.6 mL/min
   • UV detection wavelength: 254nm
   • Mobile phase: [A]: a 0.1% formic acid-containing aqueous solution, [B]: a 0.1% formic acid-containing acetonitrile solution
   • Gradient: a linear gradient of 10% to 100% solvent [B] was carried out in 3 minutes, and 100% solvent [B] was kept for 0.5 minutes.

The powder X-ray diffraction of crystals obtained in each example was measured in accordance with X-ray powder diffraction method described in the General Tests, Processes and Apparatus of the Japanese Pharmacopoeia. Measurement conditions are shown below.

• Detector: High-speed 1-dimensional detector (D/Tec Ultra 2) and variable knife edge Measurement method: Reflection method
• Type of light source: Cu tube
• Wavelength used: CuKa radiation
• Tube current: 10 mA or 15 mA
• Tube voltage: 30 Kv or 40 Kv
• Sample plate: Aluminum or glass
• X-ray incidence angle (θ): 3-40°, sampling width: 0.01° or
• X-ray incidence angle (θ): 4-40°, sampling width: 0.02°

In general, the diffraction angle (2θ) in powder X-ray diffraction may have a margin of error within the range of ±0.2°, and therefore the value of the diffraction angle also encompasses about ±0.2° range of the numerical value. Accordingly, the present invention encompasses not only a crystal, the peak diffraction angles of which in powder X-ray diffraction completely match, but also a crystal, the peak diffraction angles of which match within a margin of error of about ±0.2°.

To a solution of Compound 1 (5.0 g, 49.5 mmol) in THF (100 mL) was added dropwise 1.62mol/L n-butyllithium in hexane (30.5 mL, 49.5 mmol) at -78°C under a nitrogen atmosphere, and the mixture was stirred at -78°C for 2 hours. A solution of chloroformate allyl (5.96 g, 49.5 mmol) in THF (20 mL) was added dropwise thereto, and the mixture was stirred at -78°C for 2 hours. The mixture was quenched with a saturated aqueous solution of ammonium chloride, warmed up to room temperature, and extracted with ethyl acetate. The obtained organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain Compound 2 (5.66 g, 62%).
1H-NMR(CDCl3)δ:3.83 (t, J = 8.0Hz, 2H), 3.92 (t, J = 8.0Hz, 2H), 4.26 (s, 2H), 4.78 (d, J = 8.0Hz, 2H), 5.30 (d, J = 12.0Hz, 1H), 5.44 (d, J = 16.0Hz, 1H), 5.93-6.03 (m, 1H), Second step

To a solution of Compound 2 (6.6 g, 35.6 mmol) in THF (66 mL) was added dropwise 1.03mol/L DIBAL-H in hexane (45.0 mL, 46.3 mmol), and the mixture was stirred at -78°C for 1 hour. The mixture was quenched with acetone, an aqueous solution of Rochelle salt was added thereto. The mixture was stirred, and extracted with ethyl acetate. The obtained organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain Compound 3 (6.21 g, 93%).
1H-NMR(CDCl3)δ:3.44 (br, 1H), 3.50-3.64 (m, 2H), 3.71 (br, 1H), 3.95 (d, J = 8.0Hz, 2H), 4.64 (d, J = 8.0Hz, 2H), 5.24 (d, J = 12.0Hz, 1H), 5.40 (d, J = 16.0Hz, 1H), 5.47 (d, J = 4Hz, 1H), 5.87-6.00 (m, 1H)

To a solution of Compound 3 (6.2 g, 33.1 mmol) in methanol (65 mL) was added p-Toluenesulfonic acid monohydrate (0.63 g, 3.31 mmol), and the mixture was stirred at room temperature over night. The mixture was quenched with an aqueous solution of sodium hydrogen carbonate, concentrated, and extracted with ethyl acetate. The obtained organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain Compound 4 (5.77 g, 87%).
1H-NMR(CDCl3)δ:3.34 (s, 3H), 3.55 (br, 2H), 3.73-3.99 (m, 3H), 4.64 (d, J = 8.0Hz, 2H), 5.10-5.20 (m, 1H), 5.25 (d, J = 8.0Hz, 1H), 5.33 (d, J = 16Hz, 1H), 5.88-6.05 (m, 1H)

To a solution of Compound 5 (20.0 g, 81 mmol) in DMF (100 mL) were added ethyl iodide (22.8 g, 146 mmol) and diazabicycloundecene (18.4 mL, 122 mmol), and the mixture was stirred at room temperature over night. The mixture was poured into 10% aqueous solution of ammonium chloride, and extracted with ethyl acetate. The obtained organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain Compound 6 (22.3 g, 100%).
1H-NMR(CDCl3)δ:1.23 (t, J = 8.0Hz, 3H), 4.28 (q, J = 8.0Hz, 2H), 5.16 (s, 2H), 6.57 (d, J = 4.0Hz, 1H), 7.28-7.48 (m, 5H), 8.21 (d, J = 4.0Hz, 1H).

To a solution of Compound 6 (500 mg, 1.82 mmol) in DMA (5.0 mL) were added pyridinium p-toluenesulfonate (1.37 g, 5.47 mmol) and Boc-hydrazine (361 mg, 2.74 mmol), and the mixture was stirred at 60°C for 14 hours. To the mixture was added water and the mixture was extracted with ethyl acetate. The obtained organic layer was washed with a saturated aqueous solution of ammonium chloride and brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform-methanol) to obtain Compound 7 (519 mg, 73%).
1H-NMR(CDCl3)δ:1.24 (t, J = 8.0Hz, 3H), 1.46 (s, 9H), 4.26 (q, J = 8.0Hz, 2H), 5.28 (s, 2H), 6.40 (d, J = 8.0Hz, 1H), 7.27-7.38 (m, 4H), 7.40-7.45 (m, 2H).

Compound 7 (500 mg, 1.29 mmol) was dissolved in 4mol/L hydrogen chloride in ethyl acetate (5 mL), and the mixture was stirred at room temperature for 1 hour. The mixture was concentrated under reduced pressure. To the obtained residue was added a saturated aqueous solution of sodium hydrogen carbonate, and the mixture was extracted with dichloromethane. The obtained organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain Compound 8 (369 mg, 99%).
1H-NMR(CDCl3)δ:1.26 (t, J = 8.0Hz, 3H), 4.31 (q, J = 8.0Hz, 2H), 5.24 (s, 2H), 6.47 (d, J = 8.0, 1H), 7.28-7.44 (m, 5H), 7.64 (d, J = 8.0, 1H).

To a solution of Compound 8 (365 mg, 1.27 mmol) and Compound 4 (306 mg, 1.52 mmol) in acetonitrile (8 mL) was added dropwise tin chloride (0.223 mL, 1.90 mmol) at -25°C under a nitrogen atmosphere, and the mixture was stirred at -25°C for 45 minutes. The mixture was quenched with a saturated aqueous solution of sodium hydrogen carbonate, and dichloromethane was added thereto. The mixture was stirred at room temperature and filtered through Celite, and filtrate was extracted with dichloromethane. The obtained organic layer was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain crude Compound 9. The obtained Compound 9 was dissolved in THF (8 mL), morpholine (1.10 mL, 12.7 mmol) and tetrakis(triphenylphosphine)palladium (146 mg, 0.127 mmol) were added thereto, and the mixture was stirred at room temperature for 2 hours. To the mixture was added diethyl ether (16 mL), and the precipitated solid was filtered and dried to obtain Compound 10 (418 mg, 100%). 1H-NMR(CDCl3)δ:2.90-2.99 (m, 1H), 3.13 (t, J = 12.0Hz, 1H), 3.40-3.46 (m, 1H), 4.00-4.08 (m, 1H), 4.14 (d, J = 12.0Hz, 1H), 5.07 (s, 2H), 6.22 (d, J = 8.0Hz, 1H), 7.29-7.40 (m, 3H), 7.56 (d, J = 8.0Hz, 2H), 7.71 (d, J = 8.0Hz, 1H)

To a suspension of (R)-2-Tetrahydrofurioic Acid (855 mg, 7.36 mmol) and Compound 10 (2.00 g, 6.11 mmol) in ethyl acetate (9 ml) were added pyridine (4.00 ml, 49.6 mmol) and T3P (50% in ethyl acetate, 11.0 ml, 18.5 mmol) at room temperature, and the mixture was stirred over night. The precipitated solid was filtered and washed with ethyl acetate (4 ml) and ethanol (4 ml). The obtained solid was suspended in ethanol (6 ml) and the suspension was stirred at room temperature for 6.5 hours. The suspension was filtered and the obtained solid was washed with ethanol (2 ml) twice to obtain Compound 11 (1.18 g, 45.4%).
¹H-NMR (DMSO)δ: 1.80-1.94(m, 2H), 1.95-2.14(m, 2H), 3.21-3.35-(m, 2H), 3.50-3.60(m, 1H), 3.70-3.82(m, 3H), 4.00-4.05(m, 1H), 4.32-4.38(m, 1H), 5.14(dd, J=10.8Hz, 21.6Hz, 2H), 5.76-5.81(m, 1H), 6.29(d; J=4.8Hz, 1H), 7.28-7.39(m, 3H), 7.48-7.54(m, 2H), 7.64-7.75(m, 1H)

To a suspension of Compound 11 (500 mg, 1.18 mmol) in ethanol (3.5 ml) was added DBU (0.0035 ml, 0.023 mmol) at room temperature, and the mixture was stirred for 30 minutes. To the obtained suspension was added diisopropylether (6.5ml), and the mixture was stirred at room temperature for 30 minutes. The precipitated solid was filtered and washed with ethyl acetate (1.5 ml) twice to obtain Compound i1 (346 mg, 89.9%).
¹H-NMR (DMSO)δ: 2.80-3.00(m, 1H), 3.10-3.18(m, 1H), 3.38-3.50(m, 1H), 3.98-4.08(m, 2H), 4.10-4.20(m, 1H), 4.76-4.84(m, 1H), 5.04-5.14(m, 2H), 6.22(m, J=7.6Hz, 1H), 7.27-7.40(m, 4H), 7.56-7.60(m, 2H), 7.70(d, J=7.6Hz, 1H)

Compound i1 (1100 g, 3360 mmol) and 7,8-difluoro-6,11-dihydrodibenzothiepine-11-ol (977 g, 3697 mmol) were suspended in 50wt% T3P in ethyl acetate (3208 g, 5041 mmol) and ethyl acetate (1.1 L). To the mixture was added methanesulfonic acid (436 ml, 6721 mmol) at room temperature and the mixture was stirred at 70°C for 5.5 hours. To the mixture was added water under ice-water bath and the mixture was stirred at room temperature for 1 hour. THF was added thereto and the mixture was extracted with ethyl acetate. The obtained organic layer was washed with water and 8% aqueous solution of sodium hydrogen carbonate, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was dissolved in THF (5.5 L) and potassium carbonate (790 g, 5713 mmol) was added thereto. The mixture was warmed up to 50°C, benzyl bromide (240 ml, 2016 mmol) was added dropwise thereto, and the mixture was stirred at 60°C for 8.5 hours. To the mixture was added dropwise 2mol/L aqueous solution of hydrochloric acid under ice-water bath, and the mixture was stirred at room temperature for 10 minutes and extracted with ethyl acetate. The obtained organic layer was washed with water and 8% aqueous solution of sodium hydrogen carbonate and dried over anhydrous magnesium sulfate. An activated carbon (Norit SX-2, 240 g) was added thereto, the mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure. To the obtained residue was added ethyl acetate and hexane and the precipitated solid was filtered to obtain Compound 25 (1019 g, 1776 mmol, 53%).
¹H-NMR (CDCl₃)δ: 2.88 (1H, t, J = 11.2 Hz), 3.28-3.39 (2H, m), 3.72 (1H, d, J = 12.6 Hz), 3.86 (1H, d, J = 9.6 Hz), 4.03 (1H, d, J = 13.9 Hz), 4.45 (1H, d, J = 8.6 Hz), 4.67 (1H, d, J = 13.1 Hz), 5.19-5.26 (2H, m), 5.45 (1H, d, J = 10.9 Hz), 5.63 (1H, d, J = 10.9 Hz), 5.77 (1H, d, J = 7.6 Hz), 6.40 (1H, d, J = 7.8 Hz), 6.68 (1H, t, J = 6.9 Hz), 6.94-7.01 (2H, m), 7.03-7.12 (3H, m), 7.29-7.38 (3H, m), 7.61 (2H, d, J = 7.1 Hz).

To a solution of Compound 25 (1200 g, 2092 mmol) in DMA (3.6 L) was added lithium chloride (443g, 10.5 mol) at room temperature, and the mixture was stirred at 80°C for 3 hours. To the mixture were added acetone (1.2L), 0.5mol/L aqueous solution of hydrochloric acid (6.0 L) and water (2.4 L) under ice-water bath, and the mixture was stirred for 1 hour. The precipitated solid was filtered. The obtained solid was dissolved in chloroform, isopropyl ether was added thereto, and the precipitated solid was filtered to obtain Compound III-2 (950 g, 1965 mmol, 94%). ¹H-NMR (CDCl₃)δ: 2.99 (1H, dt, J = 17.5, 6.8 Hz), 3.47 (1H, td, J = 11.9, 2.5 Hz), 3.60 (1H, t, J = 10.6 Hz), 3.81 (1H, dd, J = 11.9, 3.3 Hz), 3.96 (1H, dd, J = 11.0, 2.9 Hz), 4.07 (1H, d, J = 13.8 Hz), 4.58 (1H, dd, J = 10.0, 2.9 Hz), 4.67 (1H, dd, J = 13.5, 1.9 Hz), 5.26-5.30 (2H, m), 5.75 (1H, d, J = 7.8 Hz), 6.69 (1H, d, J = 7.7 Hz), 6.83-6.87 (1H, m), 6.99-7.04 (2H, m), 7.07-7.15 (3H, m).

To Compound III-2 (4.0 g, 8.3 mmol) were added potassium carbonate (1483.4 mg, 10.7 mmol), potassium iodide (549.5 mg, 3.3 mmol), tetrahydrofuran (33.1 g), N,N-dimethylacetamide (3.8 g), and water (80.3 mg), and the mixture was stirred. The temperature was increased to 60°C, and chloromethyl methyl carbonate (1758.9 mg, 14.2 mmol) was added. The mixture was stirred at 60°C for 9 hours and cooled to 20°C. Acetic acid (822.0 mg), 2-propanol (3.1 g), and water (20.0 g) were added, and the mixture was extracted twice with tetrahydrofuran (1.8 g, 8.9 g). The obtained organic layer was concentrated under reduced pressure to a liquid weight of about 32 g by distilling off the solvent. The temperature was increased to 45°C, 2-propanol (1.6 g) was added, and the mixture was cooled to 20°C. An aqueous solution of sodium acetate prepared from sodium acetate (339.0 mg) and water (46.0 g) was added, and the mixture was cooled to 5°C. The mixture was stirred at 5°C for 3 hours, and the produced pale yellowish white precipitates were filtered. The obtained solids were washed with a mixed solution of 2-propanol (4.7 g) and water (6.0 g), and the solids were washed with 2-propanol (6.3 g) again. Dimethylsulfoxide (30.9 g) was added to the obtained pale yellowish white solids, and the mixture was stirred. The temperature was increased to 60°C, and a mixed solution of dimethylsulfoxide (2.2 g) and water (4.8 g) was added. Further, a mixed solution of dimethylsulfoxide (19.9 g) and water (28.4 g) was added, and the mixture was cooled to 20°C. The mixture was stirred at 20°C for 3 hours, and the produced white precipitates were filtered. The obtained solids were washed with a mixed solution of dimethylsulfoxide (8.0 g) and water (4.8 g), and the solids were washed with water (12.0 g) again. The obtained solids were dried to obtain white crystals (I-form) of Compound II-6 (4.21 g).
¹H-NMR (DMSO-D6) δ: 2.91-2.98 (1H, m), 3.24-3.31 (1H, m), 3.44 (1H, t, J = 10.4 Hz), 3.69 (1H, dd, J = 11.5, 2.8 Hz), 3.73 (3H, s), 4.00 (1H, dd, J = 10.8, 2.9 Hz), 4.06 (1H, d, J = 14.3 Hz), 4.40 (1H, d, J = 11.8 Hz), 4.45 (1H, dd, J = 9.9, 2.9 Hz), 5.42 (1H, dd, J = 14.4, 1.8 Hz), 5.67 (1H, d, J = 6.5 Hz), 5.72-5.75 (3H, m), 6.83-6.87 (1H, m), 7.01 (1H, d, J = 6.9 Hz), 7.09 (1H, dd, J = 8.0, 1.1 Hz), 7.14-7.18 (1H, m), 7.23 (1H, d, J = 7.8 Hz), 7.37-7.44 (2H, m).
Powder X-ray diffraction 2θ(°): 8.6±0.2°, 14.1±0.2°, 17.4±0.2°, 20.0±0.2°, 24.0±0.2°, 26.3±0.2°, 29.6±0.2°, and 35.4±0.2°.

Figure 3 shows powder X-ray diffraction results of I-form crystals of Compound II-6.

Acetonitrile (50 mL) and water (5 mL) were added to Compound II-6 (10.00 g), the compound was heated to dissolve, and water (95 mL) was added thereto. The solution was stirred for 10 minutes at room temperature, and the precipitated crystals were filtered. The obtained crystals were subjected to through-flow drying to obtain II-form crystals (9.04 g) of Compound II-6.

Figure 4 shows powder X-ray diffraction results of II-form crystals of Compound II-6.

Methyl acetate (400 mL) was added to Compound II-6 (10.00 g), and the compound was heated to dissolve. Methyl acetate (about 230 mL) of the solution was concentrated under reduced pressure, the solution was stirred for 70 minutes at room temperature, and the precipitated crystals were filtered. The obtained crystals were subjected to through-flow drying to obtain III-form crystals (7.87 g) of Compound II-6.

Figure 5 shows powder X-ray diffraction results of III-form crystals of Compound II-6.

Biological test examples for compounds used in the present invention were described below.

30merRNA(5'-pp-[m2'-O]GAA UAU(-Cy3) GCA UCA CUA GUAAGC UUU GCU CUA-BHQ2-3': manufactured by Japan Bio Services Co., LTD.) in which G at a 5' end is diphosphate-modified, a hydroxy group at 2' position is methoxylation-modified, U sixth from a 5' end is labelled with Cy3, and a 3' end is labelled with BHQ2 was purchased, and a cap structure was added using ScriptCap system manufactured by EPICENTRE (a product was m7G [5']-ppp-[5'] [m2'-O]GAA UAU(-Cy3) GCA UCA CUA GUAAGC UUU GCU CUA(-BHQ2)-3'). This was separated and purified by denatured polyacrylamide gel electrophoresis, and used as a substrate.

RNP was prepared from a virus particle using standard method (Reference Document: VIROLOGY(1976) 73, p327-338 OLGA M. ROCHOVANSKY). Specifically, A/WSN/33 virus (1 × 10³ PFU/mL, 200 µL) was inoculated in a 10 days old embryonated chicken egg. After incubation at 37°C for 2 days, the allantoic fluid of the chicken egg was recovered. A virus particle was purified by ultracentrifugation using 20% sucrose, solubilized using TritonX-100 and lysolecithin, and an RNP fraction (50-70% glycerol fraction) was collected by ultracentrifugation using a 30-70% glycerol density gradient, and was used as an enzyme solution (containing approximately 1 nM PB1-PB2-PA complex).

An enzymatic reaction solution (2.5 µL) (composition: 53 mM Tris-hydrochloride (pH 7.8), 1 mM MgCl₂, 1.25 mM dithiothreitol, 80 mM NaCl, 12.5% glycerol, enzyme solution 0.15 µL) was dispensed into a 384-well plate made of polypropylene. Then, 0.5 pL of a test compound solution which had been serially diluted with dimethyl sulfoxide (DMSO) was added to the plate. As a positive control (PC) or a negative control (NC), 0.5 pL of DMSO was added to the plate respectively. Each plate was mixed well. Then, 2 pL of a substrate solution (1.4 nM substrate RNA, 0.05% Tween20) was added to initiate a reaction. After room temperature incubation for 60 minutes, 1 pL of the reaction solution was collected and added to 10 pL of a Hi-Di formamide solution (containing GeneScan 120 Liz Size Standard as a sizing marker: manufactured by Applied Biosystems (ABI)) in order to stop the reaction. For NC, the reaction was stopped in advance by adding EDTA (4.5 mM) before initiation of the reaction (all concentrations described above are final concentrations).

The solution for which the reaction was stopped was heated at 85°C for 5 minutes, rapidly cooled on ice for 2 minutes, and analyzed with an ABI PRIZM 3730 genetic analyzer. A peak of the cap-dependent endonuclease product was quantitated by analysis software ABI Genemapper, a CEN reaction inhibition ratio (%) of a test compound was obtained by setting fluorescent intensities of PC and NC to be 0% inhibition and 100% inhibition, respectively, an IC₅₀ value was obtained using curve fitting software (XLfit2.0: Model 205 (manufactured by IDBS) etc.).

• 2% FCS E-MEM (prepared by adding kanamycin and FCS to MEM (Minimum Essential Medium) (Invitrogen))
• 0.5% BSA E-MEM (prepared by adding kanamycin and BSA to MEM (Minimum Essential Medium) (Invitrogen))
• HBSS (Hanks' Balanced Salt Solution)
• MDBK cell

Cells were adjusted to the appropriate cell number (3 × 10⁵/mL) with 2% FCS E-MEM.

• MDCK cell

After washing with HBSS two times, cells were adjusted to the appropriate cell number (5 × 10⁵/mL) with 0.5% BSA E-MEM.

• Trypsin solution

Trypsin from porcine pancreas (SIGMA) was dissolved in PBS(-), and filtrated with a 0.45 µm filter.

• EnVision (PerkinElmer)
• WST-8 Kit (Kishida Chemical Co., Ltd.)
• 10% SDS solution

As a culture medium, 2% FCS E-MEM was used at the use of MDBK cells, and 0.5% BSA E-MEM was used at the use of MDCK cells. Hereinafter, for diluting virus, cells and a test sample, the same culture medium was used.

A test sample was diluted with a culture medium to an appropriate concentration in advance, and then 2 to 5-fold serial dilution on a 96 well plate (50 pL/well) was prepared. Two plates, one for measuring anti-Flu activity and the another for measuring cytotoxity, were prepared. Each assay was performed triplicate for each drug.

At the use of MDCK cells, Trypsin was added to the cells to be a final concentration of 3 pg/mL only for measuring anti-Flu activity.

An influenza virus was diluted with a culture medium to an appropriate concentration in advance, and each 50 pL/well was dispensed on a 96-well plate containing a test substance. Each 50 pL/well of a culture medium was dispensed on a plate containing a test substance for measuring cytotoxity.

Each 100 pL/well of cells which had been adjusted to the appropriate cell number was dispensed on a 96 well plate containing a test sample.

This was mixed with a plate mixer, and incubated in a CO2 incubator for 3 days for measuring anti-Flu activity and measuring cytotoxity.

The cells in the 96-well plate which had been incubated for 3 days was observed visually under a microscope, and appearance of the cells, the presence or absence of a crystal of test substance were checked. The supernatant was removed so that the cells were not absorbed from the plate.

WST-8 Kit was diluted 10-fold with a culture medium, and each 100 µL was dispensed into each well. After mixing with a plate mixer, cells were incubated in a CO2 incubator for 1 to 3 hours.

After incubation, regarding the plate for measuring anti-Flu activity, each 10 pL/well of a 10% SDS solution was dispensed in order to inactivate a virus.

After the 96-well plate was mixed, absorbance was measured with EnVision at two wavelengths of 450 nm/620 nm.

The value was calculated using Microsoft Excel or a program having the equivalent calculation and processing ability, based on the following calculation equation.

$$\mathrm{EC50}={10}^{\mathrm{Z}}$$ Z = (50% - High %) / (High % - Low %) x {log(High conc.) - log(Low conc.)} + log(High conc.)

Based on the above results, the parent compounds used in the present invention exhibit high cap-dependent endonuclease (CEN) inhibitory activity and/or high CPE inhibitory effect and thus can be a useful agent for treatment and/or prevention of symptom and/or disease induced by infection with influenza virus.

Using commercially available pooled human hepatic microsome, and employing, as markers, 7-ethoxyresorufin O-deethylation (CYP1A2), tolbutamide methyl-hydroxylation (CYP2C9), mephenytoin 4'-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), and terfenedine hydroxylation (CYP3A4) as typical substrate metabolism reactions of human main five CYP enzyme forms (CYP1A2, 2C9, 2C19, 2D6, 3A4), an inhibitory degree of each metabolite production amount by a compound used in the present invention was assessed.

The reaction conditions were as follows: substrate, 0.5 pmol/L ethoxyresorufin (CYP1A2), 100 pmol/L tolbutamide (CYP2C9), 50 pmol/L S-mephenytoin (CYP2C19), 5 µmol/L dextromethorphan (CYP2D6), 1 pmol/L terfenedine (CYP3A4); reaction time, 15 minutes; reaction temperature, 37°C; enzyme, pooled human hepatic microsome 0.2 mg protein/mL; concentration of a compound used in the present invention, 1, 5, 10, 20 µmol/L (four points).

Each five kinds of substrates, human hepatic microsome, or a compound used in the present invention in 50 mmol/L Hepes buffer as a reaction solution was added to a 96-well plate at the composition as described above, NADPH, as a cofactor was added to initiate metabolism reactions as markers and, after the incubation at 37°C for 15 minutes, a methanol/acetonitrile = 1/1 (v/v) solution was added to stop the reaction. After the centrifugation at 3000 rpm for 15 minutes, resorufin (CYP1A2 metabolite) in the supernatant was quantified by a fluorescent multilabel counter and toltributamide hydroxide (CYP2C9P metabolite), mephenytoin 4' hydroxide (CYP2C19 metabolite), dextromethorphan (CYP2D6 metabolite), and terfenadine alcohol (CYP3A4 metabolite) were quantified by LC/MS/MS.

Addition of only DMSO being a solvent dissolving a compound used in the present invention to a reaction system was adopted as a control (100%), remaining activity (%) was calculated at each concentration of a compound used in the present invention added as the solution and IC50 was calculated by reverse presumption by a logistic model using a concentration and an inhibition rate.

1. (1) Experimental animals: mice or SD rats were used.
2. (2) Rearing condition: mice or SD rats were allowed free access to solid feed and sterilized tap water.
3. (3) Setting of dosage and grouping: Oral administration and intravenous administration were performed with the predetermined dosage. Grouping was set as below. (Dosage was changed per compound)
   • Oral administration 1 to 30 mg/kg (n= 2 to 3)
   • Intravenous administration 0.5 to 10 mg/kg (n= 2 to 3)
4. (4) Preparation of administration solutions: Oral administration was performed as solution or suspension. Intravenous administration was performed after solubilization.
5. (5) Routes of administration: Oral administration was performed mandatory into the stomach by oral sonde. Intravenous administration was performed from caudal vein by syringes with needle.
6. (6) Evaluation items: Blood was collected serially and concentration of a compound used in the present invention in plasma was measured by LC/MS/MS.
7. (7) Statistical analysis: About transition of concentration of a compound used in the present invention in plasma, the area under the plasma concentration versus time curve (AUC) was calculated by non-linear least-squares method program, WinNonlin (a registered trademark), and bioavailability (BA) of a compound used in the present invention was calculated from AUCs of the oral administration group and the intravenous administration group.

Based on the above results, the prodrug had improved bioavailability other than the parent compound.

Therefore, the compound used in the present invention has excellent oral absorbability and can be a useful agent for treatment and/or prevention of symptom and/or disease induced by infection with influenza virus.

Using commercially available pooled human hepatic microsomes, a compound used in the present invention was reacted for a constant time, and a remaining rate was calculated by comparing a reacted sample and an unreacted sample, thereby, a degree of metabolism in liver was assessed.

A reaction was performed (oxidative reaction) at 37 °C for 0 minute or 30 minutes in the presence of 1 mmol/L NADPH in 0.2 mL of a buffer (50 mmol/L Tris-HCl pH 7.4, 150 mmol/L potassium chloride, 10 mmol/L magnesium chloride) containing 0.5 mg protein/mL of human liver microsomes. After the reaction, 50 pL of the reaction solution was added to 100 pL of a methanol/acetonitrile = 1/1 (v/v), mixed and centrifuged at 3000 rpm for 15 minutes. The compound used in the present invention in the supernatant was quantified by LC/MS/MS or Solid Phase Extraction (SPE)/MS, and a remaining amount of the compound used in the present invention after the reaction was calculated, letting a compound amount at 0 minute reaction time to be 100%. Hydrolysis reaction was performed in the absence of NADPH and glucuronidation reaction was in the presence of 5 mM UDP-glucuronic acid in place of NADPH, followed by similar operations.
(Result) % inhibition was shown at 2pmol/L of test compound.

The CYP3A4 fluorescent MBI test is a test of investigating enhancement of CYP3A4 inhibition of a compound used in the present invention by a metabolism reaction, and the test was performed using, as CYP3A4 enzyme expressed in Escherichia coli and employing, as an index, a reaction in which 7-benzyloxytrifluoromethylcoumarin (7-BFC) is debenzylated by the CYP3A4 enzyme to produce a metabolite, 7-hydroxytrifluoromethylcoumarin (HFC) emitting fluorescent light.

The reaction conditions were as follows: substrate, 5.6 µmol/L 7-BFC; pre-reaction time, 0 or 30 minutes; reaction time, 15 minutes; reaction temperature, 25°C (room temperature); CYP3A4 content (expressed in Escherichia coli), at pre-reaction 62.5 pmol/mL, at reaction 6.25 pmol/mL (at 10-fold dilution); test drug concentration of a compound used in the present invention, 0.625, 1.25, 2.5, 5, 10, 20 µmol/L (six points).

An enzyme in a K-Pi buffer (pH 7.4) and a solution of a compound used in the present invention as a pre-reaction solution were added to a 96-well plate at the above composition of the pre-reaction, a part of it was transferred to another 96-well plate so that it was 1/10 diluted with a substrate and a K-Pi buffer, NADPH as a cofactor was added to initiate a reaction as an index (without preincubation) and, after a predetermined time of a reaction, acetonitrile/0.5 mol/L Tris (trishydroxyaminomethane) = 4/1 (V/V) was added to stop the reaction. In addition, NADPH was added to a remaining preincubation solution to initiate a preincubation (with preincubation) and, after a predetermined time of a preincubation, a part was transferred to another plate so that it was 1/10 diluted with a substrate and a K-Pi buffer to initiate a reaction as an index. After a predetermined time of a reaction, acetonitrile/0.5 mol/L Tris (trishydroxyaminomethane) = 4/1 (V/V) was added to stop the reaction. For the plate on which each index reaction had been performed, a fluorescent value of 7-HFC which is a metabolite was measured with a fluorescent plate reader. (Ex = 420 nm, Em = 535 nm).

Addition of only DMSO which is a solvent dissolving a compound used in the present invention to a reaction system was adopted as a control (100 %), remaining activity (%) was calculated at each concentration of a compound used in the present invention added as the solution, and IC₅₀ was calculated by reverse-presumption by a logistic model using a concentration and an inhibition rate. When a difference between IC₅₀ values is 5 pmol/L or more, this was defined as (+) and, when the difference is 3 pmol/L or less, this was defined as (-).

Mutagenicity of compounds used in the present invention was evaluated.

20 pL of freezing-stored rat typhoid bacillus (Salmonella typhimurium TA98 strain, TA100 strain) was inoculated on 10 mL of a liquid nutrient medium (2.5% Oxoid nutrient broth No.2), and this was cultured before shaking at 37°C for 10 hours. 9 mL of a bacterial solution of the TA98 strain was centrifuged (2000 × g, 10 minutes) to remove a culturing solution. The bacteria was suspended in 9 mL of a Micro F buffer (K₂HPO4: 3.5 g/L, KH₂PO4: 1 g/L, (NH₄)₂SO₄: 1 g/L, trisodium citrate dehydrate: 0.25 g/L, MgSO₄ • ₇H₂O: 0.1 g/L), the suspension was added to 110 mL of an Exposure medium (Micro F buffer containing Biotin: 8 pg/mL, histidine: 0.2 pg/mL, glucose: 8 mg/mL). The TA100 strain was added to 120 mL of the Exposure medium relative to 3.16 mL of the bacterial solution to prepare a test bacterial solution. Each 12 µL of DMSO solution of a compound used in the present invention (several stage dilution from maximum dose 50 mg/mL at 2 to 3 fold ratio), DMSO as a negative control, and 50 pg/mL of 4-nitroquinoline-1-oxide DMSO solution for the TA98 strain, 0.25 pg/mL of 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide DMSO solution for the TA100 strain under the non-metabolism activating condition, 40 pg/mL of 2-aminoanthracene DMSO solution for the TA98 strain, 20 µg/mL of 2-aminoanthracene DMSO solution for the TA100 strain under the metabolism activating condition as a positive control, and 588 pL of the test bacterial solution (a mixed solution of 498 µl of the test bacterial solution and 90 pL of S9 mix under the metabolism activating condition) were mixed, and this was shaking-cultured at 37°C for 90 minutes. 460 pL of the bacterial solution exposed to a compound used in the present invention was mixed with 2300 pL of an Indicator medium (Micro F buffer containing biotin: 8 pg/mL, histidine: 0.2 pg/mL, glucose: 8 mg/mL, Bromo Cresol Purple: 37.5 pg/mL), each 50 pL was dispensed into microplate 48 wells/dose, and this was subjected to stationary culturing at 37°C for 3 days. Since a well containing a bacterium which has obtained the proliferation ability by mutation of an amino acid (histidine) synthesizing enzyme gene turns from purple to yellow due to a pH change, the bacterium proliferation well which has turned to yellow in 48 wells per dose is counted, and was assessed by comparing with a negative control group. (-) means that mutagenicity is negative and (+) is positive.

For the purpose of assessing risk of an electrocardiogram QT interval prolongation of the compound used in the present invention, effects of the compound used in the present invention on delayed rectifier K+ current (I_Kr), which plays an important role in the ventricular repolarization process, was studied using HEK293 cells expressing human ether-a-go-go related gene (hERG) channel.

After a cell was retained at a membrane potential of -80 mV by whole cell patch clamp method using an automated patch clamp system (PatchXpress 7000A, Axon Instruments Inc.), I_Kr induced by depolarization pulse stimulation at +40 mV for 2 seconds and, further, repolarization pulse stimulation at -50 mV for 2 seconds, was recorded. After the generated current was stabilized, extracellular solution (NaCl: 135 mmol/L, KCI: 5.4 mmol/L, NaH₂PO4: 0.3 mmol/L, CaCl₂ • 2H₂O: 1.8 mmol/L, MgCl₂ • 6H₂O: 1 mmol/L, glucose: 10 mmol/L, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid): 10 mmol/L, pH=7.4), in which the compound used in the present invention had been dissolved at an objective concentration, was applied to the cell at room temperature for 10 minutes. From the recording I_Kr, an absolute value of the tail peak current was measured based on the current value at the resting membrane potential using analysis software (DataXpress ver.1, Molecular Devices Corporation). Further, the % inhibition relative to the tail peak current before application of the compound used in the present invention was calculated, and compared with the vehicle-applied group (0.1% dimethyl sulfoxide solution) to assess influence of the compound used in the present invention on I_Kr.
(Result) % inhibition was shown at 0.3 to 10 µM of test compound.

The solubility of the compound used in the present invention was determined under 1% DMSO addition conditions. A 10 mmol/L solution of the compound was prepared with DMSO, and 2 pL of the solution of the compound used in the present invention was added, respectively, to 198 pL of JP-1 solution (water were added to 2.0 g of sodium chloride and 7.0 mL of hydrochloric acid to reach 1000 mL) and JP-2 solution (1 volume of water were added to 1 volume of the solution which 3.40 g of potassium dihydrogen phosphate and 3.55 g of anhydrous disodium hydrogen phosphate to reach 1000 mL). The mixture was shaken for 1 hour at a room temperature, and the mixture was filtered. The filtrate was ten-fold diluted with methanol/water = 1/1(v/v), and the compound concentration in the filtrate was measured with LC/MS or SPE/MS by the absolute calibration method.

Appropriate amounts of the compound used in the present invention was put into vials and 200pL of JP-1st Fluid (water was added to 2.0g of sodium chloride in 7.0mL of hydrochloride acid to reach 1000 mL), JP-2nd Fluid (water was added to 500 mL of phosphate buffer solution with a pH of 6.8) and 20mmol /L sodium taurocholate (TCA) / JP-2nd Fluid (JP-2nd Fluid was added to 1.08g of TCA in JP-2nd Fluid to reach 100mL) was added to each vial. When the compound was completely dissolved, appropriate amount of compound was added. After shaken for 1 hour at 37°C, the mixture was filtered and 100µL of methanol was added to 100µL of each filtrate (double dilution). Dilution magnification was changed if necessary. After it was confirmed whether there were air bubbles and precipitates in the vials, the vials were shaken with tight stopper. The compound concentration was determined with HPLC by the absolute calibration method.

Ames test was performed by using Salmonellas (Salmonella typhimurium) TA 98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA as test strains with or without metabolic activation in the pre-incubation method to check the presence or absence of gene mutagenicity of compounds used in the present invention.

The compound used in the present invention was dissolved at target concentrations and was mixed with a 2.5 v/v% suspension of red blood cells prepared from a defibrinated blood of sheep on a microplate at concentrations of 0.0008 to 0.1 w/v%. The mixtures were exposed to 10°J/cm² of UV-irradiation within a range of wavelength 290 to 400 nm, UVA and UVB using ultra violet fluorescent lamps, GL20SE and FL20S-BLB lamps manufactured by Sankyo Denki Co., Ltd. and Panasonic Corporation, respectively. After the completion of the irradiation, the mixtures were centrifuged, and a supernatant of the mixture was collected and was located on a microplate. The phototoxicity was assessed by measuring an absorbance at wavelength of 540 nm and 630 nm in the supernatant. The absorbance data at wavelength of 540 nm and 630 nm were used as indicators of biomembrane damage (photohemolysis %) and hyperoxidation of lipid membrane (methemoglobin formation), respectively. The criteria of phototoxicity was as follows; It was judged to be non-phototoxic (-) when the photohemolysis % < 10 and the maximal change in the absorbance at 630 nm (ΔOD) < 0.05 were observed. It was judged to be non-phototoxic (+) when the photohemolysis was more than 10% and the maximal change in the absorbance at 630 nm (ΔOD) was more than 0.05.

The plasma concentration of Compound III-2 and Compound II-6 after oral administration of prodrug Compound II-6, the parent compound of which was Compound III-2, to rat under non-fasting conditions was measured. The result is shown in Figures 1 and 2.

The concentration of Compound II-6 in all plasma samples was a determination limit or less. Therefore, prodrug Compound II-6, the parent compound of which was Compound III-2 was found to have changed promptly to Compound III-2 in vivo after administration (see Figure 2).

Based on the above test results, it was revealed that the compound converted into a prodrug was absorbed into the body after oral administration, and rapidly converted into a parent compound in the blood. Therefore, the compounds (parent compounds and/or prodrugs) used in the present invention can be useful agents for treatment and/or prevention of symptoms and/or diseases induced by infection with influenza virus.

1. (1) Animals used: SD rats were used.
2. (2) Rearing conditions: Pellets and sterilized tap water were fed to SD rats ad libitum.
3. (3) Dosage and grouping: A predetermined dosage was intravenously administered. Groups were set as follows. (Dosage varied for each compound)
   Intravenous administration 0.5-1 mg/kg (n = 2-3)
4. (4) Preparation of administration solution: Intravenous administration was performed after solubilization.
5. (5) Administration method: Intravenous administration was performed with a needle-equipped syringe on the caudal vein.
6. (6) End point: Blood was collected over time, and the plasma concentration of the compound used in the present invention was measured using LC/MS/MS.
7. (7) Statistical analysis: As for the transition of the plasma concentration of the compound used in the present invention, the total body clearance (CLtot) and the elimination half-life (t1/2, z) were calculated using nonlinear least-squares program WinNonlin (Registered trademark).

From the above results, it was found that Compound III-2 is a compound having a low total body clearance and a long half-life.

Therefore, the compound used in the present invention has excellent persistence and can be a useful agent for treatment and/or prevention of symptom and/or disease induced by infection with influenza virus.

The efficacy and safety of a single oral administration of an investigational drug (active ingredient (Compound II-6): 10 mg, 20 mg, 40 mg) to patients infected by influenza virus were evaluated by a randomized, placebo-controlled, double-blind comparative study. As for the primary endpoint, subjects made evaluations by themselves on a 4-point scale [0: none, 1: mild, 2: moderate, 3: severe] concerning the time to alleviation of influenza symptoms (the time from the beginning of administration of the investigational drug until 7 influenza symptoms ("cough", "sore throat", "headache", "nasal congestion", "feverishness or chills", "muscular or joint pain", and "fatigue") were alleviated) to evaluate the efficacy of the investigational drug over the placebo.
Patients who satisfied all of the following criteria were selected as subjects.

• (a) Male or female patients at 20 years old or older and younger than 65 years old
• (b) Patients satisfying all of the following criteria and diagnosed with influenza virus infectious disease
  • Positive in influenza rapid diagnosis [Rapid antigen test (RAT)] based on a nasal or throat swab
  • Body temperature (axillary temperature) of 38.0°C or higher
  • Having one or more moderate or severer symptoms among the following systemic symptoms and respiratory symptoms due to influenza virus infectious disease
    • Systemic symptoms (headache, feverishness or chills, muscular or joint pain, fatigue)
    • Respiratory symptoms (cough, sore throat, nasal congestion)
• (C) Patients within 48 hours from onset (at registration)

The definition of onset is any of the following.

• When the body temperature increased for the first time (at least an increase of 1°C from normal temperature)
• When any one or more of the systemic symptoms and respiratory symptoms were developed

• 10 mg Tablet of Compound II-6: White to pale yellowish white, circular, film-coated tablet containing 10 mg of Compound II-6
• 20 mg Tablet of Compound II-6: White to pale yellowish white, elliptical, film-coated tablet containing 20 mg of Compound II-6

• Placebo for 10 mg tablet of Compound II-6: Tablet undistinguishable from 10 mg tablet of Compound II-6
• Placebo for 20 mg tablet of Compound II-6: Tablet undistinguishable from 20 mg tablet of Compound II-6

Eligible subjects were randomly allocated to a Compound II-6 10 mg group, 20 mg group, 40 mg group, and placebo group in a ratio of 1:1:1:1. Subjects received a single oral administration of total 3 tables of Compound II-6 tablets and/or placebo tablets in a combination indicated in the following table on Day 1.

Treatment Groups Cormpound II-6 10 mg tablet Compound II-6 20 mg tablet Placebo tablet Matching Compound II-6 10 mg tablet Placebo tablet Matching Compound II-6 20 mg tablet Compound II-6 10 mg tablet 1 tablet - - 2 tablets Compound II-6 20 mg tablet - 1 tablet 1 tablet 1 tablet Compound II-6 40 mg tablet - 2 tablets 1 tablet - Placebo - - 1 tablet 2 tablets

The main efficacy endpoint is the time to alleviation of influenza symptoms (the time to alleviation of influenza symptoms).

It is the time from the beginning of administration until alleviation of influenza symptoms. Alleviation of influenza symptoms refers to when all 7 influenza symptoms (cough, sore throat, headache, nasal congestion, feverishness or chills, muscular or joint pain, fatigue) become "0: none" or "1: mild" in the patient diary that the subject keeps, and this condition continues at least 21.5 hours (24 hours - 10%).

The secondary efficacy endpoint is as follows.

It is the time from the beginning of administration until alleviation of each influenza symptom. Alleviation of a symptom refers to when the target item becomes "0: none" or "1: mild", and this condition continues at least 21.5 hours (24 hours - 10%).

As for the time to alleviation of influenza symptoms, which is the primary endpoint, the primary analysis and the secondary analysis are described. In addition to the ITTI group, the primary analysis was also performed on the PPS group for sensitivity analysis. Other analyses were performed only on the ITTI group.

The hazard ratio, 95% confidence interval, and P value of each administered group relative to the placebo group were calculated by a Cox proportional hazard model using the time to alleviation of influenza symptoms as a response, the administered groups as fixed effects, and the current smoking habit and the total score of 7 influenza symptoms at baseline before administration, which are allocation factors, as covariates. In order to prevent an increase of the probability of type I error due to performing the test multiple times, the P value was adjusted by the Hommel's method.

The placebo group and each investigational drug administered group were compared by stratified generalized Wilcoxon test using the time to alleviation of influenza symptoms as a response, the administered groups as explanatory variables, and the category (11 points or less, 12 points or more) of the total score of 7 influenza symptoms before administration and the smoking habit, which are allocation factors, as stratification factors.

Also, a Kaplan-Meier survival curve was drawn for each group to calculate the median time to alleviation of influenza symptoms and the 95% confidence interval thereof. The Greenwood's method was used for calculating the confidence interval.

The same analysis as in the primary endpoint was performed, with the time until each alleviation of influenza symptom being regarded as a response. At this time, cases where the symptom before administration was "0: none" or "1: mild" were excluded from the analysis target.

Out of 400 randomly selected patients, 389 patients (98 patients (98%) in the 10 mg administered group, 95 patients (95%) in the 20 mg administered group, 99 patients (99%) in the 40 mg administered group, and 97 patients (97%) in the placebo group) completed the test. As for the primary endpoint, the ITTI Population (cases where an investigational drug was administered, and influenza virus infection was confirmed) consisted of 400 patients.

The per protocol set cases consisted of 368 patients (89 patients (89%) in the 10 mg administered group, 92 patients (92%) in the 20 mg administered group, 96 patients (96%) in the 40 mg administered group, and 91 patients (91%) in the placebo group). As for the ITTI Population of each group, it was found from the rapid antigen detection test that 75% to 79% of the patients were infected by influenza A virus, and 21% to 25% of the patients were infected by influenza B virus.

Analysis results are shown in the following tables. Testdrug 10 mg administered group Testdrug 20 mg administered group Test drug 40 mg administered group Placebo admnistered group N (human) 100 100 100 100 Median value (hour) 54.2 51 49.5 77.7 95% Confidence interval (hour) 47.7,66.8 44.5, 62.4 44.5, 64.4 67.6, 88.7 Difference from placebo (hour) -23.4 26.6 28.2 - Generalized Wilson test P value 0.0085 0.0182 0.0046 - Cox proportional hazard model relative to placebo Hazard ratio 0.758 0.81 0.817 - 95% Confidence interval 0.571, 1.007 0.608, 1.078 0.614, 1.087 - P value 0.0561 0.1488 0.165 -

The primary endpoint of this test, i.e., the median time until the symptoms were alleviated, was 54.2 hours in the 10 mg administered group (95% CI: 47.7, 66.8), 51.0 hours in the 20 mg administered group (95% CI: 47.7, 66.8), 49.5 hours in the 40 mg administered group (95% CI: 44.5, 64.4), and 77.7 hours in the placebo group (95% CI: 67.6, 88.7).

The following tables show the results of analyzing the time until each of the 7 influenza symptoms ("cough", "sore throat", "headache", "nasal congestion", "feverishness or chills", "muscular or joint pain", "fatigue") is alleviated.

Testdrug 10 mg administered group Testdrug 20 mg administered group Testdrug 40 mg administered group Placebo administered group N (human) 49 38 45 47 Median value (95% CI) (hour) 25.2 (19.0, 47.2) 21.6 (13.4, 30.5) 21.9 (16.0, 28.7) 42.8 (22.9, 68.3) Difference from placebo (hour) 17.6 21.3 21 - P value (G. Wilcoxon test ^a 0.043 0.0516 0.0003 - Hazard ratio (95% CI)^b 0.742 (0.494, 1.114) 0.59 0.379, 0.920) 0.564 (0.369, 0.862) - P value (Cox model)^b 0.15 0.0199 0.0081 -

Test drug 10 mg administered group Testdrug 20 mg administered group Testdrug 40 mg administered group Placebo administered group N (human) 73 77 71 71 Median value (95% CI) (hour) 31.2 (24.9, 39.9) 29.9 (22.8, 37.0) 25.4 (20.5, 28.9) 41.9 (28.7,48.6) Difference from placebo (hour) -10.7 12 16.4 - P value (G. Wilcoxon test ^a 0.2153 0.0346 0.0048 - Hazard ratio (95% CI)^b 0.77 (0.553, 1.072) 0.687 (0.494, 0.955) 0.657 (0.469, 0.920) - P value (Cox model)^b 0.1217 0.0255 0.0145 -

Testdrug 10 mg administered group Testdrug 20 mg administered group Testdrug 40 mg administered group Placebo administered group N (human) 82 82 77 79 Median value (95% CI) (hour) 32 (29.2, 39.9) 31.3 (26.7, 42.4) 31.1 (24.6,38.6) 42.7 (30.3,53.2) Difference from placebo (hour) -10.7 -11.5 -11.7 - P value (G. W ilcoxon test)^a 0.1221 0.0594 0.0224 - Hazard ratio (95% CI)^b 0.783 (0.574, 1.069) 0.8/6 0.637, 1.203) 0.724 (0.527, 0.995) - P value (Cox model)^b 0.1236 0.412 0.0463 -

Testdrug 10 mg administered group Testdrug 20 mg administered group Test drug 40 mg administered group Placebo administered group N (human) 97 93 94 95 Median value (95% CI) (hour) 24.7 (21.3, 28.4) 29.4 (22.0, 34.8) 23 (19.8, 28.6) 28.8 (21.1, 33.4) Difference from placebo (hour) -4.1 0.6 -5.8 - P value (G. Wilcoxon test)^a 0.0602 0.3774 0.0258 - Hazard ratio (95% CI)^b 0.635 (0.475, 0.850) 0.848 0.634, 1.133) 0.71 (0.529, 0.951) - P value (Cox model)^b 0.0023 0.2642 0.0216 -

Testdrug 10 mg administered group Testdrug 20 mg administered group Testdrug 40 mg administered group Placebo administered group N (human) 61 58 54 57 Median value (95% CI) (hour) 42.2 (29.8, 47.3) 37 (28.5, 43.5) 37.9 (28.6, 44.5) 43.7 (29.7, 53.6) Difference from placebo (hour) -1.5 -6.7 -5.8 - P value (G. W ilcoxon test ^a 0.6846 0.7741 0.0904 - Hazard ratio (95% CI)^b 0.803 (0.557, 1.157) 0.936 0.635, 1.381) 0.655 (0.447, 0.961) - P value (Cox model)^b 0.2388 0.7404 0.0304 -

Testdrug 10 mg administered group Testdrug 20 mg administered group Test drug 40 mg administered group Placebo administered group N (hum an) 74 74 78 75 Median value (95% CI) (hour) 31.1 (21.3,41.5) 29.8 (21.9, 32.9) 24.6 (16.1, 29.4) 31.2 (20.9, 51.4) Difference from placebo (hour) -0.1 -1.4 -6.6 - P value (G. Wilcoxon test ^a 0.6643 0.8536 0.1551 - Hazard ratio (95% CI)^b 0.941 (0.675, 1.312) 0.883 0.636, 1.226) 0.865 (0.626, 1.196) - P value (Cox model)^b 0.7188 0.4569 0.3796 -

Testdrug 10 mg administered group Testdrug 20 mg administered group Test drug 40 mg administered group Placebo administered group N (hum an) 56 64 55 46 Median value (95% CI) (hour) 35.3 (21.2, 49.8) 2/.8 (19.9, 32.1) 31.9 (17.3, 43.0) 26.3 (16.5, 45.2) Difference from placebo (hour) 9.1 1.5 5.6 - P value (G. W ilcoxon test ^a 0.2905 0.6293 0.993 - Hazard ratio (95% CI)^b 1.312 (0.882, 1.951) 1.05 0.713, 1.547) 1.092 (0.738, 1.617) - P value (Cox model)^b 0.18 0.8047 0.6602 - a Stratified generalized Wilson test relative to placebo. Stratification factors: Smoking habit, and composite symptom scores at baseline.
b Cox proportional hazard model relative to placebo. Covariates: Smoking habit, and composite symptom scores at baseline.
Subset of patients whose score of symptoms at baseline was "moderate" or "severe" CI: Confidence interval

An analysis using a Cox proportional hazard model revealed that the 40 mg administered group in comparison to the placebo group showed a significant decrease in time until the following 5 symptoms: "nasal congestion", "muscular or joint pain", "fatigue", "feverishness or chills", and "headache" were alleviated. For example, as for 2 symptoms, i.e., "nasal congestion" and "muscular or joint pain", the median times until these symptoms were improved were 21.0 hours and 16.4 hours, respectively, and they were shorter in the 40 mg administered group than the placebo group.

Statistically significant differences were observed also in the 10 mg administered group and the 20 mg administered group with respect to the following symptoms: "muscular or joint pain", "nasal congestion", and "feverishness or chills".

The efficacy and safety of a single oral administration of an investigational drug (active ingredient (Compound II-6): 40 mg, 80 mg) to patients infected by influenza virus were evaluated by a randomized, double-blind comparative study in comparison to 75 mg Oseltamivir administered twice per day for 5 days or a placebo. As for the primary endpoint, subjects made evaluations by themselves on a 4-point scale [0: none, 1: mild, 2: moderate, 3: severe] concerning the time to alleviation of influenza symptoms (the time from the beginning of administration of the investigational drug until 7 influenza symptoms ("cough", "sore throat", "headache", "nasal congestion", "feverishness or chills", "muscular or joint pain", and "fatigue") were alleviated) to evaluate the efficacy of the investigational drug over the placebo.

Moreover, as for the secondary efficacy endpoint, the efficacy and the side effects of the investigational drug were evaluated according to the influenza virus titer using a nasal or throat swab.
Patients who satisfied all of the following criteria were selected as subjects.

1. (a) Male or female patients at 12 years old or older and younger than 65 years old
2. (b) Patients satisfying all of the following criteria and diagnosed with influenza virus infectious disease
   • Body temperature (axillary temperature) of 38.0°C or higher
   • Having one or more moderate or severer symptoms among the following systemic symptoms and respiratory symptoms due to influenza virus infectious disease
     • Systemic symptoms (headache, feverishness or chills, muscular or joint pain, fatigue)
     • Respiratory symptoms (cough, sore throat, nasal congestion)
3. (c) Patients within 48 hours from onset (at registration)

The definition of onset is any of the following.

• When the body temperature increased for the first time (at least an increase of 1°C from normal temperature)
• When any one or more of the systemic symptoms and respiratory symptoms were developed

Method for administering investigational drug

20 mg Tablet of Compound II-6

• Placebo for 20 mg tablet of Compound II-6
• 75 mg Capsule of Oseltamivir
• Placebo for 75 mg capsule of Oseltamivir: Capsule undistinguishable from 75 mg capsule of Oseltamivir

Eligible patients at 20 to 64 years old were randomly allocated to a group receiving a single administration of Compound II-6 (40 or 80 mg depending on the body weight), a group receiving 75 mg Oseltamivir twice a day for 5 days, and a placebo group in a ratio of 2:2:1.

Eligible patients at 12 to 19 years old were randomly allocated to a group receiving a single administration of Compound II-6 (40 or 80 mg depending on the body weight) and a placebo administered group in a ratio of 2:1.

The dosage of Compound II-6 was 40 mg for subjects weighing less than 80 kg, and 80 mg for subjects weighing 80 kg or more.

20 mg Tablets of Compound II-6 were administered orally (2 tablets or 4 tablets depending on the body weight). Placebo capsules for Oseltamivir were administered orally twice a day (morning, evening), one capsule per administration.

Placebo capsules for Oseltamivir were administered orally twice a day (morning, evening), one capsule per administration.

Placebo tablets for Compound II-6 were administered orally (2 tablets or 4 tablets depending on the body weight). 75 mg Capsules of Oseltamivir were administered orally twice a day (morning, evening), one capsule per administration.

75 mg Capsules of Oseltamivir were administered orally twice a day (morning, evening), one capsule per administration.

Placebo tablets for Compound II-6 were administered orally (2 tablets or 4 tablets depending on the body weight). Placebo capsules for Oseltamivir were administered orally twice a day (morning, evening), one capsule per administration.

Placebo capsules for Oseltamivir were administered orally twice a day (morning, evening), one capsule per administration.

"Day 1" indicates the first day of administration, and "Day 2 to Day 5" indicates the second day to the fifth day as counted from the first day of administration.

The main efficacy endpoint is the time to alleviation of influenza symptoms (the time to alleviation of influenza symptoms).

It is the time from the beginning of administration until alleviation of influenza symptoms. Alleviation of influenza symptoms refers to when all 7 influenza symptoms (cough, sore throat, headache, nasal congestion, feverishness or chills, muscular or joint pain, fatigue) become "0: none" or "1: mild" in the patient diary that the subject keeps, and this condition continues at least 21.5 hours (24 hours - 10%).

The secondary efficacy endpoint is as follows.

1. (1) Proportion of patients having a positive influenza virus titer at each point
2. (2) Amount of change in virus titer from baseline at each point
3. (3) Time to termination of viral shedding based on virus titer
4. (4) Incidence of side effects

The virus titer was measured in the following manner.

1. (1) MDCK-SIAT1 cells seeded in a flat-bottom 96-well microplate were cultured in a 5% CO₂ incubator at 37±1°C for 1 day.
2. (2) A standard strain (influenza virus AH3N2, A/Victoria/361/2011, storage condition: -80°C, origin: National Institute of Infectious Diseases), a sample (collected from patients in Phase III clinical test of Compound II-6 and stored in an ultra-low-temperature freezer), and a medium for cell control were diluted 101 to 107 folds by a 10-fold serial dilution method.
3. (3) After cells present in a sheet form were confirmed under an inverted microscope, the medium was removed, and a new medium was added at 100 pL/well.
4. (4) The medium was removed.
5. (5) Each of the samples (100 to 107) prepared at (2) above was inoculated at 100 pL/well, using 4 wells per sample.
6. (6) Centrifugal adsorption was performed at room temperature at 1000 rpm for 30 minutes.
7. (7) After centrifugation, the medium was removed, and cells were washed once with a new medium.
8. (8) A new medium was added at 100 pL/well.
9. (9) Incubation was performed in a 5% CO₂ incubator at 33±1°C for 3 days.
10. (10) After incubation, the CytoPathic Effect (CPE) was evaluated under an inverted microscope.

When the detection limit was exceeded as measured by the above virus titer measurement method, it was determined to be positive.

As for the time to alleviation of influenza symptoms, which is the primary endpoint, the primary analysis and the secondary analysis are described. The primary analysis was performed on the ITTI group.

For patients at 12 to 64 years old, the placebo group and the investigational drug administered group were compared by stratified generalized Wilcoxon test using the total score of 7 influenza symptoms before administration (11 points or less, 12 points or more) and regions (Japan/Asia, other regions) as stratification factors.

Also, a Kaplan-Meier survival curve was drawn for each group to calculate the median time to alleviation of influenza symptoms and the 95% confidence interval thereof as well as the difference between the groups in the time to alleviation of influenza symptoms and the 95% confidence interval thereof.

For patients at 20 to 64 years old, the time to alleviation of influenza symptoms was compared between the Compound II-6 group and the Oseltamivir group by the same method as the primary analysis.

The following secondary efficacy endpoints were compared between the Compound II-6 group and the placebo group and between the Compound II-6 group and the Oseltamivir group (the age group of 20 to 64 years old).

Only the patients having a virus titer equal to or greater than the determination limit before the beginning of administration in Visit 1 were included in the analysis. In each Visit, a Mantel-Haenszel test using the total score of 7 influenza symptoms before administration and the regions as stratification factors was applied, and the proportion of patients having a positive virus titer was compared between two groups.

Only the patients having a virus titer before the beginning of administration in Visit 1 were included in the analysis. In each Visit, a van Elteren test using the total score of 7 influenza symptoms before administration and the regions as stratification factors was applied, and the amount of change in influenza virus titer from the baseline was compared between two groups.

Only the patients having a virus titer equal to or greater than the determination limit before the beginning of administration in Visit 1 were included in the analysis. A stratified generalized Wilcoxon test using the total score of 7 influenza symptoms before administration and the regions as stratification factors was applied.

The number of side-effect episodes and the number of patients with side effect were counted for each administration group.

Out of 1436 randomly selected patients, 1366 patients (578 patients in the 40 mg or 80 mg Compound II-6 administered group, 498 patients in the Oseltamivir administered group, and 290 patients in the placebo group) completed the test. As for the primary endpoint, the ITTI cases (cases where GCP was followed, the investigational drug was administered, and influenza virus infection was confirmed) consisted of 1064 patients.

The per protocol set cases consisted of 990 patients (427 patients in the 40 mg or 80 mg Compound II-6 administered group, 351 patients in the Oseltamivir administered group, and 212 patients in the placebo group).

Analysis results are shown in the following table. 12 Years old or older and younger than 65 years old 20 Years old or older and younger than 65 years old Compound II-6 administered group Placebo administered group Compound II-6 administered group Oseltamivir administered group Number of patients 455 230 375 377 Median (hour) 53.7 80.2 53.5 53.8 95% Confidence interval (hour) 49.5, 58.5 72.6, 87.1 48.0, 58.5 50.2, 56.4 Difference between groups^a (hour) -26.5 --- -0.3 --- 95% Confidence interval of difference between groups (hour)^b -35.8, .17.8 --- -6.6, 6.6 --- Stratified generalized Wilcoxon test^c p Value a <.0001 --- 0.7560 --- a vs Placebo or vs Oseltamivir
b Bootstrap estimation
c Used the regions and the total score of 7 influenza symptoms before administration as stratification factors, and censored at final evaluation for patients whose symptoms were no alleviated.

In the ITTI group, the time to alleviation of influenza symptoms (median) (95% CI) was 53.7 hours (95% CI: 49.5, 58.5) in the Compound II-6 group while 80.2 hours (95% CI: 72.6, 87.1) in the placebo group, and the difference between the Compound II-6 group and the placebo group was -26.5 hours. The time to alleviation of influenza symptoms of the Compound II-6 group was significantly shorter than that of the placebo group in the primary analysis using a stratified generalized Wilcoxon test (p<.0001).

In the subgroup of patients at 20 years old or older and younger than 65 years old, the time to alleviation of influenza symptoms was 53.5 hours (95% CI: 48.0, 58.5) in the Compound II-6 group while 53.8 hours (95% CI: 50.2, 56.4) in the Oseltamivir group, and the difference between the Compound II-6 group and the Oseltamivir group was -0.3 hours. There was no significant difference between the times to alleviation of influenza symptoms of the Compound II-6 group and the Oseltamivir group in the stratified generalized Wilcoxon test.

Analysis results are shown in the following table. Observation time point 12 Years old or older and younger than 65 years old 20 Years old or older and younger than 65 years old Compound II-6 administered group Placebo administered group Compound II-6 administered group Oseltamivir administered group N=427 N=210 N=352 N=359 Day 2 Proportion 47.8% (197/412) 96.0% (1931201) 47.6% (161/338) 91.0% (315/346) 95% Confidence interval 42.9, 52.8 92.3, 98.3 42.2, 53.1 87.5, 93.8 p Value ^a <.0001 --- <.0001 --- Day 3 Proportion 21.5% (67/404) 70.2% (1341191) 19.8% (661333) 57.3% (197/344) 95% Confidence interval 17.6, 25.9 63.1, 76.5 15.7, 24.5 51.9, 62.6 p Value ^a <.0001 --- <.0001 --- Day 4 Proportion 16.7% (191114) 56.1% (32157) 16.1% (14/87) 27.6% (291105) 95% Confidence interval 10.3, 24.8 42.4, 69.3 9.1,25.5 19.3, 37.2 p Value^a <.0001 --- 0.0852 --- Day 3 Proportion 13.6% (55/403) 29.7% (57/192) 13.0% (431339) 20.9% (70/335) 95% Confidence interval 10.4, 17.4 23.3, 36.7 9.6, 17.1 16.7, 25.6 p Value ^a <.0001 --- 0.0066 --- Day 6 Proportion 8.2% (8197) 12.5% (6148) 5.6% (4171) 9.0% (7/78) 95% Confidence interval 3.6, 15.6 4.7,25.2 1.6, 13.8 3.7, 17.6 p Value ^a 0.4767 --- 0.6187 --- Day 9 Proportion 2.9% (12/407) 4.6% (91197) 3.0% (101335) 3.2% (111339) 95% Confidence interval 1.5, 5.1 2.1, 8.5 1.4, 5.4 1.6, 5.7 p Value ^a 0.3379 --- 0.8618 --- Day 2 indicates 24 hours later, as counted from the first day of administration, Day 3 indicates 48 hours later, Day 4 indicates 72 hours later, Day 5 indicates 96 hours later, Day 6 indicates 120 hours later, and Day 9 indicates 192 hours later.
a vs Placebo or vs Oseltamivir. Mantel-Haenszel test. Used the regions and the total score of 7 influenza symptoms before administration as stratification factors, and intended for a group having a positive virus titer before administration.

The proportion of patients having a positive virus titer was significantly lower in the Compound II-6 group than in the placebo group on Day 2 (Mantel- Haenszel test: p<.0001), and likewise, significantly lower in the Compound II-6 group than in the placebo group on Day 3 (p<.0001). In the subgroup of patients at 20 years old or older and younger than 65 years old, the proportion of patients having a positive virus titer was significantly lower in the Compound II-6 group than in the Oseltamivir group on Day 2 and Day 3 (p<.0001).

Analysis results are shown in the following table. Observation time point 12 Years old or older and younger than 65 years old 20 Years old or older and younger than 65 years old Compound II-6 administered group Placebo administered group Compound II-6 administered group Oseltamivir administered group N=427 N=210 N=352 N=359 Day 2 Number of patients 412 201 338 346 Mean -4.44 -1.19 -4.39 -2.51 Standard deviation 2.03 2.43 2.07 2.03 p Value ^a <.0001 --- <,0001 --- Day 3 Number of patients 404 191 333 344 Mean -4.82 -2.91 -4.78 -4.20 Standard deviation 1.99 2.85 2.03 2.02 p Value ^a <.0001 --- <.0001 --- Day 4 Number of patients 114 57 87 105 Mean -4.50 -3.31 -4.46 -4.63 Standard deviation 2.02 2.34 2.03 1.89 p Value ^a 0.0008 --- 0.8010 --- Day 5 Number of patients 403 192 331 335 Mean -4.95 -4.47 -4.95 -4.98 Standard deviation 1.93 2.21 1.94 1.82 p Value ^a 0.0132 --- 0.9425 --- Day 6 Number of patients 97 48 71 78 Mean -4.58 -4.68 -4.56 -4.85 Standard deviation 1.99 2.12 1.99 1.95 p Value ^a 0.9307 --- 0.2256 --- Day 9 Number of patients 407 197 335 339 Mean -5.06 -4.87 -5.03 -5.22 Standard deviation 1.87 1.85 1.89 1.70 p Value ^a 0.1684 --- 0.3267 --- Unit: logic [TCID₅₀/mL].
Day 2 indicates 24 hours later, as counted from the first day of administration, Day 3 indicates 48 hours later, Day 4 indicates 72 hours later, Day 5 indicates 96 hours later, Day 6 indicates 120 hours later, and Day 9 indicates 192 hours later.
a vs Placebo or vs Oseltamivir. van Elteren test. Used the regions and the total score of 7 influenza symptoms before administration as stratification factors. Intended for a group having a positive virus titer before administration.

The virus titer decreased significantly in the Compound II-6 group as compared to the placebo group on Day 2, and likewise, decreased significantly as compared to the placebo group on Day 3 (van Elteren test: p<.0001). In the subgroup of patients at 20 years old or older and younger than 65 years old, the virus titer decreased significantly in the Compound II-6 group as compared to the Oseltamivir group on Day 2 and Day 3 (p<.0001).

Analysis results are shown in the following table. 12 Years old or older and younger than 65 years old 20 Years old or older and younger than 65 years old Compound II-6 administered group Placebo administered group Compound II-6 administered group Oseltamivir administered group Number of patients 95% Confidence interval (hour) 423 207 346 355 24.0, 48.0 --- 24.0, 48.0 72.0, 96.0 Difference between groups (hour)^a -72.0 --- -48.0 --- Stratified generalized Wilcoxon test^b p Value <.0001 --- <.0001 --- a vs Placebo or vs Oseltamivir.
b Used the regions and the total score of 7 influenza symptoms before administration as stratification factors.
Censored at final evaluation for patients whose virus titer was not eliminated.
Intended for analyzing patients who had a positive virus titer on Day 1 and whose data concerning the time to termination of viral shedding was not missing.

The time (median) to termination of viral shedding based on virus titer was 24.0 hours in the Compound II-6 group while 96.0 hours in the placebo group, and was significantly shorter in the Compound II-6 group than in the placebo group (stratified generalized Wilcoxon test: p<.0001). The time to termination of viral shedding in the subgroup of patients at 20 years old or older and younger than 65 years old was 24.0 hours in the Compound II-6 group and 72.0 hours in the Oseltamivir group, and was significantly shorter in the Compound II-6 group than in the Oseltamivir group (p<.0001).

Severe adverse events the causal relationship of which cannot be denied are not reported. Adverse events the causal relationship of which cannot be denied occurred in 27 patients out of 610 patients (4.4%, 37 episodes) in the Compound II-6 group, 12 patients out of 309 patients (3.9%, 19 episodes) in the placebo group, and 43 patients out of 513 patients (8.4%, 53 episodes) in the Oseltamivir group. There was no statistically significant difference between the incidences in the Compound II-6 group and the placebo group (Fisher's exact test, two-sided P value: 0.8627). However, the incidence in the Compound II-6 group was significantly lower than that in the Oseltamivir group (Fisher's exact test, two-sided P value: 0.0088).

The efficacy and safety of a single oral administration of an investigational drug (active ingredient (Compound II-6): 5 mg, 10 mg, 20 mg, 40 mg) to patients infected by influenza virus were evaluated. As for the primary endpoint, guardians or subjects by themselves made evaluations and measurements concerning the time to alleviation of influenza symptoms (the time from the beginning of administration of the investigational drug until influenza symptoms ("cough", "runny nose/nasal congestion", and "fever") were alleviated) to evaluate the efficacy of the investigational drug.

"Cough" and "runny nose/nasal congestion" were evaluated on a 4-point scale [0: none, 1: mild, 2: moderate, 3: severe].

Patients who satisfied all of the following criteria were selected as subjects.

1. (a) Male or female patients at 6 months old or older and younger than 12 years old
2. (b) Patients satisfying all of the following criteria and diagnosed with influenza virus infectious disease
   • Positive in influenza rapid diagnosis [Rapid antigen test (RAT)] based on a nasal or throat swab
   • Body temperature (axillary temperature) of 38.0°C or higher
   • Having one or more moderate or severer symptoms among the respiratory symptoms due to influenza virus infectious disease for patients at 7 years old or older
3. (c) Patients within 48 hours from onset (at registration). The onset is defined as when the body temperature exceeding 37.5°C is confirmed for the first time.
4. (d) Patients having a body weight of 5 kg or more.

• 5 mg Tablet of Compound II-6: Half of 10 mg tablet of Compound II-6
• 10 mg Tablet of Compound II-6
• 20 mg Tablet of Compound II-6

Patients received a single oral administration on Day 1 in a dose calculated based on the body weight (see the table below). Body weight of patient at the time of screening Dose of Compound II-6 Compound II-6 tablet 5 kg or more and less than 10 kg 5 mg Half of 10 mg tablet 10 kg or more and less than 20 kg 10 mg One 10 mg tablet 20 kg or more and less than 40 kg 20 mg One 20 mg tablet or two 10 mg tablets 40 kg or more 40 mg Two 20 mg tablets

The main efficacy endpoint is the time to alleviation of influenza symptoms (the time to alleviation of influenza symptoms).

It is the time from the beginning of administration until alleviation of influenza symptoms. Alleviation of an influenza symptom refers to the time when a and b below are satisfied from the beginning of administration, and this clinical condition continues at least 21.5 hours (24 hours - 10%).

1. a. "Cough" and "runny nose/nasal congestion" are both "0: none" or "1: mild" in the patient diary
2. b. Body temperature (axillary temperature) is lower than 37.5°C

As for the time to alleviation of influenza symptoms, which is the primary endpoint, the primary analysis is described. The primary analysis was performed on the ITTI group.

A Kaplan-Meier curve of the time to alleviation of influenza symptoms ("cough", "runny nose/nasal congestion", and "fever") (the time to alleviation of influenza symptoms) was drawn to calculate the median time to complete alleviation of influenza symptoms and the 95% confidence interval thereof. Patients whose influenza symptoms were not completely alleviated during the observation period were treated as censored cases.

As for the primary endpoint, 103 patients were involved. The time (median) to alleviation of influenza symptoms in the ITTI group was 44.6 hours (95% CI: 38.9, 62.5).

The following Formulation Examples are only exemplified and not intended to limit the scope of the invention.

The compounds used in the present invention, lactose and calcium stearate are mixed. The mixture is crushed, granulated and dried to give a suitable size of granules. Next, calcium stearate is added to the granules, and the mixture is compressed and molded to give tablets.

The compounds used in the present invention, lactose and calcium stearate are mixed uniformly to obtain powder medicines in the form of powders or fine granules. The powder medicines are filled into capsule containers to give capsules.

The compounds used in the present invention, lactose and calcium stearate are mixed uniformly and the mixture is compressed and molded. Then, it is crushed, granulated and sieved to give suitable sizes of granules.

The compounds used in the present invention and crystalline cellulose are mixed, granulated and tablets are made to give orally disintegrated tablets.

The compounds used in the present invention and lactose are mixed, crushed, granulated and sieved to give suitable sizes of dry syrups.

The compounds used in the present invention and phosphate buffer are mixed to give injection.

The compounds used in the present invention and phosphate buffer are mixed to give injection.

The compound used in the present invention and lactose are mixed and crushed finely to give inhalations.

The compounds used in the present invention and petrolatum are mixed to give ointments.

The compounds used in the present invention and base such as adhesive plaster or the like are mixed to give patches.

The parent compounds used in the present invention have cap-dependent endonuclease (CEN) inhibitory activity after absorption into the body. The compounds (the parent compounds and/or the prodrugs) used in the present invention can be useful agents for treatment and/or prevention of symptom and/or disease induced by infection with influenza virus. The pharmaceutical composition is effective for shortening the time to alleviation of influenza symptoms, and is useful for treating and/or preventing an influenza virus infectious disease.